Adjuvant and vaccine compositions containing monophosphoryl lipid A

ABSTRACT

The invention pertains to adjuvant and vaccine compositions of monophosphoryl lipid A, sugar and optionally an amine based surfactant, which when frozen and thawed or lyophilized and reconstituted reform a colloidal suspension having a light transmission of greater than or equal to 88% as measured spectrophotometrically.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 09/352,526 filed on Jul.13, 1999 now U.S. Pat. No. 6,306,404, the entire disclosure of which isherein incorporated by reference, which application claims the benefitof U.S. patent application Ser. No. 09/115,392 filed Jul. 14, 1998,which was converted to a Provisional Application herein incorporated byreference pursuant to a petition filed under 37 C.F.R. 1.53 (c) (2)filed Nov. 4, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to improved adjuvant and vaccine compositions,methods for preparing said improved adjuvant and vaccine compositions,and methods of using the improved compositions.

2. Description of the Prior Art

Conventional vaccines have been used for many years to protect humansand animals from a wide variety of infectious diseases. Typically, theseconventional vaccines contain one or more antigens which may include anattenuated pathogen, killed pathogen, or an immunogenic component of apathogen. In some vaccines, the antigen or antigens may be employedalone to elicit protective immune responses. In other vaccines, theantigen or antigens may be employed together with one or more adjuvantsto enhance the immunogenicity of an antigen. One such adjuvant known tothe art is monophosphoryl lipid A, which is derived from thelipopolysaccharide of Salmonella minnesota R595. It is also known to theart that monophosphoryl lipid A is a lipidic material whichspontaneously aggregates with itself in an aqueous environment.Moreover, it is known that the degree of aggregation has an effect onthe activity of monophosphoryl lipid A as an immunostimulant in that theaggregated monophosphoryl lipid A is less stimulatory.

Monophosphoryl lipid A is typically obtained as the triethylamine saltin the form of a lyophilized white powder. Being very hydrophobic, thelyophilized monophosphoryl lipid A does not readily form a clearsolution when reconstituted with water but instead yields a turbidmixture with visible white particulates of heterogeneous size thatsettle out and further aggregate upon standing. To make an acceptableaqueous preparation of monophosphoryl lipid A, it is known to suspendthe lyophilized monophosphoryl lipid A triethylamine salt at 1 to 2mg/mL (w/v) in water containing 0.2% triethylamine, to heat thesuspension at 65-70° C., and then to sonicate the mixture. The resultingaqueous preparation, slightly opalescent or clear, is an aqueouscolloidal suspension. The triethylamine aids in the solubilization ofthe monophosphoryl lipid A and may be substituted with similar amountsof triethanolamine.

When aqueous preparations of monophosphoryl lipid A prepared asdescribed hereinabove are frozen and then thawed, however, themonophosphoryl lipid A aggregates resulting in a turbid mixture quitesimilar in appearance to the turbid mixture of monophosphoryl lipid Aprior to sonication. Similarly, when an aqueous preparation ofmonophosphoryl lipid A as described hereinabove is lyophilized and thenrehydrated, the result is also a turbid mixture of aggregatedmonophosphoryl lipid A.

SUMMARY OF THE INVENTION

The present invention provides to the art a lyophilized compositioncontaining monophosphoryl lipid A, which composition exhibits anenhanced reconstitution feature and which avoids the settling out andaggregation problems of the prior art. In particular, the presentinvention provides a lyophilized composition comprising monophosphoryllipid A, sugar and, optionally, an added amine based surfactant, and iscapable of being reconstituted or rehydrated with an aqueous diluent toform, without further sonication, an aqueous colloidal suspension ofmonophosphoryl lipid A having a light transmission of at least 88%, asmeasured spectrophotometrically. The lyophilized composition accordingto the present invention comprises up to about 5 wt % monophosphoryllipid A, greater than about 70 wt % sugar and from about 0 to about 30wt % optionally added amine based surfactant, said wt % based on thetotal of the weights of monophosphoryl lipid A, sugar and, if present,amine based surfactant. Preferably, the lyophilized compositionaccording to the present invention comprises up to about 5 wt %monophosphoryl lipid A, from about 70 to about 99.99 wt % sugar and fromabout 0 to about 28 wt % optionally added amine based surfactant. Morepreferably, the lyophilized composition according to the presentinvention comprises up to about 4 wt % monophosphoryl lipid A, fromabout 75 to about 99.99 wt % sugar and from about 0 to about 22 wt %optionally added amine based surfactant. The lyophilized composition mayfurther comprise an immunologically effective amount of an antigen orantigens. The lyophilized composition of the present invention may bereconstituted or rehydrated with an aqueous diluent at concentrations upto about 210 mg of lyophilized composition per ml of aqueous diluent,preferably from about 10 mg of lyophilized composition per ml of aqueousdiluent to about 210 mg of lyophilized composition per ml of aqueousdiluent, to form, without further sonication, an aqueous colloidalsuspension.

Another aspect of the present invention is a method of preparing anaqueous colloidal suspension of monophosphoryl lipid A in which theaqueous colloidal suspension is frozen for storage and then thawed foruse without the problems of settling out and aggregation known in theprior art. By this method, monophosphoryl lipid A is mixed in an aqueousdiluent and optionally with an amine based surfactant and alsooptionally an antigen or antigens. An aqueous colloidal suspension isformed by sonicating, optionally with heating, or other known methods,as described in greater detail hereinafter. Sugar, in an amount fromabout 10 mg/ml to about 200 mg/ml, is added to the mixture either beforeor after the formation of an aqueous colloidal suspension. The sugar maybe in the form of a solid or in the form of an aqueous solution. Theresulting aqueous colloidal suspension may then be frozen. Thawing thefrozen aqueous colloidal suspension affords without further sonicationan aqueous colloidal suspension containing monophosphoryl lipid A havinga light transmission of greater than or equal to 88%, as measuredspectrophotometrically. An antigen or antigens, as defined hereinafter,may be added to the thawed aqueous colloidal suspension to form avaccine composition which may be administered to a vertebrate.Alternatively, if the aqueous colloidal suspension contains an antigenbefore freezing, the vaccine composition may be thawed and administeredto a vertebrate.

The aqueous colloidal suspensions of the present invention are a specialtype of liquid suspension in which the particles of suspendedmonophosphoryl lipid A are present in very finely divided but not indissolved form. The aqueous colloidal suspensions containingmonophosporyl lipid A, sugar and, optionally, an amine based surfactantaccording to the present invention are true suspensions not solutions,and do not have the property, unlike ordinary suspensions ofmonophosphoryl lipid A, of settling out and aggregation. The presence ofthe aqueous colloidal suspensions of the present invention can bedetermined by light transmission. Thus, an aqueous colloidal suspensioncontaining monophosphoryl lipid A, sugar and optionally an amine basedsurfactant according to the present invention is one which exhibits alight transmission of greater than or equal to 88%, as measuredspectrophotometrically.

The present invention solves the settling out and aggregation problemsof the prior art, by providing the addition of sugar to an aqueouscolloidal suspension of monophosphoryl lipid A prior to freezing orlyophilization. The sugar may be added either before or after formationof the aqueous colloidal suspension but must be added before freezing orlyophilization of the suspension. The addition of sugar to an aqueouscolloidal suspension of monophosphoryl lipid A prior to freezing orlyophilization provides a composition which, after freezing can bethawed to afford an aqueous colloidal suspension without furthersonication or, alternatively, after lyophilization, can be reconstitutedwith a suitable aqueous diluent and afford without further sonication anaqueous colloidal suspension as described hereinabove. Suitable sugarsinclude the monosaccharides, dextrose, mannose, galactose and fructoseas well as the disaccharides sucrose, lactose, isomaltose, maltose andtrehalose. Mixtures of sugars, for example sucrose and dextrose, mayalso be employed. These sugars are all non toxic and pharmaceuticallyacceptable. Preferred are sucrose and dextrose. The sugar may be in theform of a solid or in the form of an aqueous solution. Suitable aqueousdiluents include water or saline and can also include an antigen orantigens and, may additionally contain preservatives or additionaladjuvants, or other pharmaceutically acceptable additives, vehicles, orcarriers. Suitable amine based surfactants include triethylamine (TEA)and triethanolamine (TEM).

A further aspect of the invention is a reconstituted or rehydratedaqueous colloidal suspension which, despite the elimination of a furthersonication step, is obtained upon reconstitution of the lyophilizedcomposition described hereinabove with an aqueous diluent. As discussedhereinabove, before the present invention, a sonication step wasnecessary in order to obtain an aqueous colloidal suspension containingmonophosphoryl lipid A. However, it has now been found that when anaqueous diluent is added to the lyophilized composition describedhereinabove, an aqueous colloidal suspension containing monophosphoryllipid A is obtained without further sonication. The reconstitutedaqueous colloidal suspension so obtained exhibits a light transmissionof greater than or equal to 88%, when measured spectrophotometrically.Surprisingly, the reconstituted aqueous colloidal suspension so obtainedis capable of being frozen and, after thawing, again reforming anaqueous colloidal suspension which exhibits a light transmission ofgreater than or equal to 88%. The reconstituted aqueous colloidalsuspension of the present invention comprises up to about 2.5 mg ofmonophosphoryl lipid A per ml of aqueous diluent, from about 10 to 200mg of sugar per ml of aqueous diluent, and from about 0 to about 6 mg ofamine based surfactant per ml of aqueous diluent. Preferably, thereconstituted aqueous colloidal suspension of the present inventioncomprises up to about 2.0 mg of monophosphoryl lipid A per ml of aqueousdiluent, from about 20 to 150 mg of sugar per ml of aqueous diluent andfrom about 0 to about 3 mg of amine based surfactant per ml of diluent.The reconstituted aqueous colloidal suspension may further comprise animmunologically effective amount of an antigen or antigens. Suitablesugars, amine based surfactants and aqueous diluents are as describedhereinabove.

A further aspect of the invention is a vaccine composition comprisingthe lyophilized composition and the reconstituted aqueous colloidalsuspension described hereinabove in combination with an immunologicallyeffective amount of an antigen or antigens. The effective amount of anantigen or antigens may be optionally provided in the aqueous diluent.In particular, the vaccine composition further comprises animmunologically effective amount of an antigen or antigens derived fromor produced by a bacterium, a virus, a parasite, a cancer cell or anallergen. An effective amount of antigen is defined as that amount ofantigen that when administered to an animal or a human evokes an immuneresponse as measured by production of specific antibodies orcell-mediated effector mechanisms. Immunologically effective amounts ofan antigen or antigens are in general from about 1 μg or less to 5 mg.An effective amount of the monophosphoryl lipid A adjuvant is the amountof monophosphoryl lipid A that when added to a vaccine will enhance themagnitude or quality or duration of the immune response to the antigenor antigens in the vaccine. An effective amount of the adjuvantmonophosphoryl lipid A is in the range of about 1 μg to about 1 mg.

Suitable antigens for the vaccine compositions of the present inventioninclude any entity capable of producing an antibody or cell-mediatedimmunological response directed specifically against that entity in avertebrate exposed to the antigen. One or more antigens may be employed.The antigen or antigens may be derived from pathogenic micro-organismsincluding viruses, bacteria, mycoplasmas, fungi, protozoa and otherparasites. Further, the antigen or antigens may be derived from sourcesother than microorganisms, for example, cancer cells or allergens. Theantigen or antigens may be all or part of a pathogenic microorganism, orall or part of a protein, glycoprotein, glycolipid, polysaccharide orlipopoly-saccharide which is associated with the organism, or theantigen or antigens may be a polypeptide or other entity which mimicsall or part of such a protein, glycoprotein, glycolipid, polysaccharideor lipopolysaccharide.

Pathogenic microorganisms from which antigens may be produced or derivedfor vaccine purposes are well known in the field of infectious diseases,as listed in, for example, Medical Microbiology, Second Edition, (1990)J. C. Sherris (ed.), Elsevier Science Publishing Co., Inc., New York,and Zinsser Microbiology, 20th Edition (1992), W. K. Joklik et al.(eds.), Appleton & Lange Publishing Division of Prentice Hall, EnglewoodCliffs, N.J. Examples of organisms of interest for human vaccinesinclude Chlamydia, Nontypeable Haemophilus influenzae, Helicobacterpylori, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseriameningitidis, Salmonella typhi, Streptococcus pneumoniae, Group AStreptococcus, Group B Streptococcus, Herpes Simplex Virus, HumanImmunodeficiency Virus, Human Papilloma Virus, Influenza, Measles,Parainfluenza, Respiratory Syncytial Virus, Rotavirus, Norwalk Virus,and others.

The antigen or antigens may include glycoconjugates which comprisepolysaccharide antigen or antigens, for example, bacterial capsularpolysaccharide or fragment thereof, chemically linked to a proteincarrier molecule in order to enhance immunogenicity. Methods forpreparing conjugates of bacterial capsular polysaccharide and proteincarrier molecules are well known in the art and can be found, forexample, in Dick and Burret, Contrib Microbiol Immunol. 10:48-114(CruseJ M, Lewis R E Jr., eds; Basel Kruger (1989). Suitable conjugates,including pneumococcal glycoconjugate, are described in greater detailin U.S. Pat. Nos. 4,673,574, 4,761,283, 4,902,506, 5,097,020 and5,360,897 the contents of which are incorporated herein by reference.

Also provided is a method of immunizing a vertebrate through vaccinationwhich comprises administrating an effective amount of a vaccinecomposition according to the present invention to said vertebrate.

Also provided is a method for the preparation of a lyophilizedcomposition comprising:

a. suspending monophosphoryl lipid A in an amount up to about 5 mg/mland, optionally, an amine based surfactant in an amount from 0 to about6 mg/ml in an aqueous diluent;

b. forming an aqueous colloidal suspension having a light transmissionof greater than or equal to 88%, as measured spectrophotometrically;

c. adding sugar at about 10 to 200 mg/ml either before or after formingthe aqueous colloidal suspension;

d. lyophilizing the sugar containing aqueous colloidal suspension; and

e. recovering a lyophilized composition.

Also provided is a method for preparing a lyophillized compositioncomprising:

a. heating lipopolysaccharide of gram negative bacteria Salmonellaminnesota R595 in a mineral acid of moderate strength for a sufficientperiod of time to obtain a monophosphoryl derivative;

b. dissolving the monophosphoryl derivative in an organic solvent anddrying;

c. treating the monophosphoryl derivative with mild alkali to remove abase labile fatty acid chain at the position to yield 3-deacylatedmonophosphoryl lipid A;

d. purifying the 3-deacylated monophosphoryl lipid A by liquidchromatography and recovering monophosphoryl lipid A;

e. suspending monophosphoryl lipid A in an amount up to about 5 mg/mland, optionally, an amine based surfactant in an amount from 0 to about6 mg/ml in an aqueous diluent;

f. forming an aqueous colloidal suspension having a light transmissionof greater than or equal to 88%, as measured spectrophotometrically;

g. adding sugar at about 10 to 200 mg/ml either before or after formingthe aqueous colloidal suspension;

h. lyophilizing the sugar containing aqueous colloidal suspension; and

i. recovering a lyophilized composition.

Also provided is a method for the preparation of an aqueous colloidalsuspension containing monophosphoryl lipid A capable of being frozen andthawed comprising:

a. suspending monophosphoryl lipid A in an amount up to about 5 mg/mland, optionally, an amine based surfactant in an amount from 0 to about6 mg/ml in an aqueous diluent;

b. forming an aqueous colloidal suspension having a light transmissionof greater than or equal to 88%, as measured spectrophotometrically;

c. adding sugar at about 10 to 200 mg/ml either before or after formingthe aqueous colloidal suspension;

d. freezing the sugar containing aqueous colloidal suspension; and

e. thawing and recovering the aqueous colloidal suspension.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preparation of monophosphoryl lipid A is described in U.S. Pat. No.4,912,094, the contents of which are incorporated herein by reference.Briefly, monophosphoryl lipid A is produced by refluxinglipopolysaccharide (or lipid A) obtained from heptoseless mutants ofgram negative bacteria, Salmonella minnesota R595, in mineral acidsolutions of moderate strength (e.g., 0.1N HCl) for a period ofapproximately 30 minutes. Suitable mineral acids include hydrochloric,sulfuric and the like. This treatment results in the loss of thephosphate moiety at position 1 of the reducing-end glucosamine. The corecarbohydrate is removed from the 6′ position of the non-reducingglucosamine during this treatment. The result is a monophosphorylderivative of lipid A. The monophosphoryl derivative of lipid A isdissolved in organic solvents and treated with very mild alkali whichremoves the base-labile fatty acid chain at the 3 position to yield3-O-desacyl-4′-monophosphoryl lipid A, indicating that position 3 of thereducing end glucosamine is de-O-acylated. Chemically it is a mixture of3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.Suitable organic solvents include methanol (alcohols), dimethylsulfoxide, dimethylformamide, chloroform, dichloromethane and the likeas well as mixtures thereof. Combinations of water and one or more ofthese organic solvents also can be employed. Suitable alkaline bases canbe chosen from among various hydroxides, carbonates, phosphates andamines. Illustrative bases include the inorganic bases such as sodiumhydroxide, potassium hydroxide, sodium carbonate, potassium carbonate,sodium bicarbonate, potassium bicarbonate, and the like, and organicbases such as alkyl amines and include, but are not limited to,diethylamine, triethylamine and the like. The3-O-desacyl-4′-monophosphoryl lipid A is purified by liquidchromatography and converted to the monobasic triethylamine(triethylammonium) salt.

The term monophosphoryl lipid A as used herein means3-O-desacyl-4′-monophosphoryl lipid A as the monobasic triethylamine(triethylammonium)salt.

To prepare the lyophilized composition of the present invention, themonophosphoryl lipid A is added to an aqueous diluent, preferably water,in amounts up to 5 mg of monophosphoryl lipid A per ml of aqueousdiluent, preferably up to 2.5 mg/ml and more preferably from about 0.5to 2.5 mg/ml. Optionally, an added amine based surfactetnt in an amountfrom about 0 to about 6 mg/ml, preferably 0 to 3 mg/ml is employed.

An aqueous colloidal suspension having a light transmission of greaterthan or equal to 88%, as measured spectrophotometrically is formed bysonication, optionally with heating, or other methods. Heating isoptional but preferred to facilitate the formation of the aqueouscolloidal suspension of monophosphoryl lipid A. Suitable sonicationequipment include, for example, a probe sonicator (Vibracell VCX600;Sonica) attached to probes whose sizes are appropriate for the volumebeing processed or a bath sonicator such as the Model No. G112SP1Tobtained from Laboratory Supplies Co. Inc., (Hicksville, N.Y.). Othersimilar equipment used in the pharmaceutical industry would also beappropriate for sonication of monophosphoryl lipid A.

The aqueous colloidal suspension of monophosphoryl lipid A may be formedby methods other than sonication, for example, by shearing forces aswould be generated in a microfluidizer.

Sugar is also added either before or after formation of the aqueouscolloidal suspension, in amounts from 10 to 200 mg sugar per ml ofaqueous diluent, preferably from about 20 to 150 mg/ml. The aqueouscolloidal suspension, containing monophosphoryl lipid A, sugar andoptionally an added amine based surfactant and optionally animmunologically effective amount of an antigen or antigens in theamounts recited hereinabove, is lyophilized to afford the lyophilizedcomposition according to the present invention.

The aqueous colloidal suspension of monophosphoryl lipid A, sugar and,optionally, an amine based surfactant of an antigen or antigens islyophilized to afford the lyophilized composition of the presentinvention. As is known to those skilled in the art, lyophilization is aprocess of drying in which water is sublimed from the product after itis frozen, by applying a vacuum. Specifics of lyophilizing orfreeze-drying are described in Remington's Pharmaceutical Sciences.Chapter 84, page 1565, 18th Edition, A. R. Gennaro, Editor, 1990, MackPublishing Company.

Whether an aqueous colloidal suspension is formed is determined bymeasuring the light transmission. It has been found that compositionshaving a light transmission of at least 88% exhibit the properties ofcolloidal suspensions. Light transmission is measured using aspectrophotometer in which is illuminated a liquid sample in a glass,quartz or plastic cuvette with a light path of 1 centimeter. The lightmay be in the visible or invisible spectrum, but for measurements oflight transmission of this type a wavelength of 650 nm may appropriatelybe used. The amount of light passing through the sample (i.e.transmitted) is referenced to a blank cuvette containing the solvent ordiluent in which the material is dissolved or suspended. Samples that donot absorb or scatter the light will exhibit 100% light transmissionwhereas those that absorb or scatter all the light will have 0% lighttransmission.

While not wishing to be bound by theory, it is believed that theadvantageous results of the invention are obtained because the additionof sugar either before or after the formation of an aqueous colloidalsuspension containing monophosphoryl lipid A prevents the monophosphoryllipid A from aggregation either upon freezing or thawing of the aqueouscolloidal suspension or upon lyophilizing the aqueous colloidalsuspension and reconstitution or rehydration with an aqueous diluent. Byincluding sugar in an aqueous colloidal suspension containingmonophosphoryl lipid A prior to lyophilization, the lyophilizedcomposition can be reconstituted with an aqueous diluent such as wateror saline without the problem of reaggregation of the monophosphoryllipid A. In addition, freezing of the reconstituted colloidal suspensionor vaccine composition does not cause aggregation to reoccur. Similarly,by including sugar in an aqueous colloidal suspension containingmonophosphoryl lipid A prior to freezing, upon thawing a frozen aqueouscolloidal suspension is again obtained without the need for furthersonication. The ability of sugar to prevent aggregation of themonophosphoryl lipid A is evident regardless of whether the aqueouscolloidal suspension containing monophosphoryl lipid A is prepared inwater alone, or in water containing triethylamine or triethanolamine.

Thus, the addition of sugar to monophosphoryl lipid A containing aqueouscompositions, either before or after forming an aqueous colloidalsuspension, provides surprising and unexpected results when such aqueouscolloidal suspensions are either frozen and thawed or lyophilized andreconstituted. Such results further permit the advantageous preparationof vaccine compositions.

The following examples are provided to illustrate the invention.

EXAMPLE 1 Preparation of Turbid Mixture and Measurement of LightTransmission

Monophosphoryl lipid A (RIBI ImmunoChem., Hamilton, Mont.) is suspendedin water at 1 mg/ml (w/v) forming a turbid mixture with visible whiteparticulates of heterogeneous size. The turbid mixture is placed in aShimadzu UV-1601, UV-Visible Spectrophotometer and illuminated withlight of 650 nm wavelength. The turbid mixture allows 3.3% of theincident light to pass (i.e. transmission=3.3). An aqueous colloidalsuspension is not found.

EXAMPLE 2

Preparation of Aqueous Colloidal Suspension and Measurement of LightTransmission

Monophosphoryl lipid A, at 1 mg/ml (w/v) is suspended in watercontaining 0.5% triethanolamine (v/v) (5.62 mg/mL (w/v)) or 0.2%triethylamine (v/v) (1.46 mg/mL(w/v)). The samples are heated at 56-65°C. for 10-15 minutes and sonicated using either a probe sonicator(Vibracell VCX600) set at 30% power using a tapered microtip or a bathsonicator (Model No. G112SP1T, Laboratory Supplies Co. Inc., Hicksville,N.Y.) used at full power for 2 to 3 minutes. A clear suspension isobtained and placed in a Shimadzu UV-1601, UV-Visible Spectrophotometerand illuminated with light of 650 nm wavelength. The % lighttransmission is measured at ≧88%, indicating the formation of an aqueouscolloidal suspension.

EXAMPLE 3 Preparation of Aqueous Colloidal Suspension of Monophosphoryllipid A and Lyophilization

Aqueous colloidal suspensions of monophosphoryl lipid A are formed bysuspending monophosphoryl lipid A, at 1, 2 or 5 mg/mL (w/v) in water orwater containing either 0.5% triethanolamine v/v (5.6 mg/mL w/v), or0.2% triethylamine v/v (1.46 mg/mL w/v). Each Monophosphoryl lipid Asuspension is heated for 10-15 minutes at 56° C. to 65° C. and thensonicated for a total of 2-3 minutes to obtain a clear suspension withno visual evidence of particulates. The samples (1 to 1.5 ml) aresonicated using either a probe sonicator (Vibracell VCX600) set at 30%power using a tapered microtip or a bath sonicator (Model No. G112SP1T,Laboratory Supplies Co. Inc., Hicksville, N.Y.) used at full power.Aliquots of the monophosphoryl lipid A aqueous colloidal suspensionsabove are diluted with an equal amount of water, or sucrose or dextrosesolutions of varying concentrations. The resulting aqueous colloidalsuspensions include monophosphoryl lipid A at 0.5, 1.0 or 2.5 mg/mL(w/v) and sucrose at final concentrations of 10, 50, 100 or 200 mg/ml(w/v) or dextrose at 10, 50, 100 or 170 mg/ml (w/v) as expressed inTable 1. The preparations contained either triethanolamine (TEM) at 2.81or 5.62 mg/mL or triethylamine (TEA) at 0.73 mg/mL or no amine basedsurfactant. The samples are placed in a Shimadzu UV-1601, UV-VisibleSpectrophotometer and illuminated with light of 650 nm wavelength. The %light transmission, as set forth in Table 1, ranges from 90.0 to 99.9%,indicating the formation of an aqueous colloidal suspension.

TABLE 1 COMPOSITION OF MONOPHOSPHORYL LIPID A FORMULATIONS Added MPLsugar sugar Amine Amine % light Sample mg/mL added mg/mL added mg/mLtransmission 1 0.5 sucrose 10 TEM 2.81 97.2 2 0.5 dextrose 10 TEM 2.8197.1 3 0.5 sucrose 10 TEM 2.81 97.2 4 0.5 dextrose 10 TEM 2.81 97.3 50.5 sucrose 10 TEA 0.73 98.9 6 0.5 sucrose 10 TEA 0.73 98.4 7 0.5sucrose 50 TEM 5.62 95.9 8 0.5 sucrose 50 TEM 5.62 96.0 9 0.5 sucrose 50TEM 2.81 97.5 10 0.5 dextrose 50 TEM 2.81 97.4 11 0.5 sucrose 50 TEM2.81 97.5 12 0.5 dextrose 50 TEM 2.81 97.4 13 0.5 sucrose 50 TEA 0.7398.8 14 0.5 sucrose 50 TEA 0.73 99 15 0.5 sucrose 50 — 0 96.1 16 0.5sucrose 50 — 0 96.0 17 0.5 sucrose 100 TEM 2.81 97.6 18 0.5 dextrose 100TEM 2.81 97.6 19 0.5 sucrose 100 TEM 2.81 97.4 20 0.5 dextrose 100 TEM2.81 97.6 21 0.5 dextrose 170 TEM 2.81 98.2 22 0.5 dextrose 170 TEM 2.8198.1 23 0.5 dextrose 200 TEM 2.81 98.4 24 0.5 sucrose 200 TEM 2.81 98.425 0.5 sucrose 200 TEM 2.81 97.7 26 0.5 sucrose 200 TEM 2.81 97.8 27 0.5sucrose 200 TEA 0.73 99.4 28 0.5 sucrose 200 TEA 0.73 99.4 29 0.5sucrose 200 TEA 0.73 98.9 30 0.5 sucrose 200 TEA 0.73 98.9 31 1.0sucrose 200 TEA 0.73 97.7 32 1.0 sucrose 200 TEA 0.73 97.6 33 1.0sucrose 200 TEM 2.81 95.7 34 1.0 sucrose 200 TEM 2.81 95.6 35 2.5sucrose 200 TEM 2.81 90.1 36 2.5 sucrose 200 TEM 2.81 90.0 37 2.5sucrose 200 TEA 0.73 95.4 38 2.5 sucrose 200 TEA 0.73 95.3

Lyophilization of Monophosphoryl Lipid A Adjuvant Compositions

The aqueous colloidal suspensions set forth in Table 1 are lyophilizedby first freezing the samples in glass vials or polypropylene culturetubes on dry ice pellets for at least 30 minutes. They are thentransferred to large freeze drying vessels (Labconco) and connected to aVirtus Freeze Dryer. The samples are lyophilized for 18 hours at avacuum of 250 millitors and the condenser temperature of −50° C. Thecomposition of the lyophilized adjuvant compositions are shown in Table2.

Reconstitution of Lyophilized Adjuvant Compositions

The lyophilized samples as set forth in Table 2 are reconstituted witheither water or normal saline (0.9% NaCl w/v), the volume of which wasequal to the volume of the aqueous colloidal suspension prior tolyophilization. Data showing the % light transmission of the samplesafter reconstitution with aqueous diluent are presented in Table 2. Asshown in Table 2, the lyophilized compositions, containing sucrose ordextrose ranging from greater than 75% up to 99.4% of the composition byweight, gave rise to aqueous colloidal suspensions when rehydrated withwater or saline. For samples 1-38 set forth in Table 2, the %transmission after rehydration ranged from 88.0% to 98.4% indicating theformation of an aqueous colloidal suspension. Samples 15 and 16, whichcontained 99% sugar by weight after lyophilization, were preparedwithout the addition of amines (triethylamine or triethanolamine) at thetime of sonication. When rehydrated with either water or normal saline,% transmission values are measured at 96.1 and 93.6, respectively,indicating the formation of an aqueous colloidal suspension. These datashow that when an aqueous colloidal suspension of Monophosphoryl lipid Aprepared by sonication is lyophilized with an effective amount of sugarsuch as sucrose or dextrose it can be rehydrated with water or normalsaline to regain an aqueous colloidal suspension.

TABLE 2 Light transmission properties of lyophilized MonophosphorylLipid A compositions after rehydration with water or normal saline.Composition Wt. % after lyophilization % light % transmission % % AddedDiluent for after Sample MPL sugar Amine rehydration rehydration 1 3.875.1 21.1 water 95.8 2 3.8 75.1 21.1 water 96.1 3 3.8 75.1 21.1 saline95.5 4 3.8 75.1 21.1 saline 95.4 5 4.5 89.0 6.5 water 96.5 6 4.5 89.06.5 saline 94.8 7 0.9 89.1 10 water 95.7 8 0.9 89.1 10 saline 95.7 9 0.993.8 5.3 water 96.0 10 0.9 93.8 5.3 water 96.6 11 0.9 93.8 5.3 saline95.7 12 0.9 93.8 5.3 saline 96.3 13 1.0 97.6 1.4 water 98.4 14 1.0 97.61.4 saline 96.3 15 1.0 99.0 0 water 96.1 16 1.0 99.0 0 saline 93.6 170.5 96.8 2.7 water 96.4 18 0.5 96.8 2.7 water 97.1 19 0.5 96.8 2.7saline 95.8 20 0.5 96.8 2.7 saline 96.8 21 0.3 98.1 1.6 water 97.4 220.3 98.1 1.6 saline 96.6 23 0.2 98.4 1.4 water 97.7 24 0.2 98.4 1.4saline 96.8 25 0.2 98.4 1.4 water 97.2 26 0.2 98.4 1.4 saline 96.9 270.2 99.4 0.4 water 98.4 28 0.2 99.4 0.4 saline 96.7 29 0.2 99.4 0.4water 95.5 30 0.2 99.4 0.4 saline 96.7 31 0.5 99.1 0.4 water 97.1 32 0.599.1 0.4 saline 96.1 33 0.5 98.1 1.4 water 95.0 34 0.5 98.1 1.4 saline94.6 35 1.2 97.4 1.4 water 89.8 36 1.2 97.4 1.4 saline 88.0 37 1.2 98.40.4 water 94.8 38 1.2 98.4 0.4 saline 90.8

EXAMPLE 4

Using the procedures set forth in Example 3, formulations containingmonophosphoryl lipid A, sugar and amine in the amounts set forth inTable 3 are prepared. The light transmission of these formulations ismeasured and as set forth in Table 3, % light transmission ranges from95.4 to 98.8% indicating the formation of an aqueous colloidalsuspension.

TABLE 3 COMPOSITION OF MONOPHOSPHORYL LIPID A FORMULATIONS Added MPLsugar sugar Amine Amine % Light Sample mg/mL added mg/mL added mg/mLTransmission 39 0.5 — 0 TEM 5.62 95.8 40 0.5 — 0 TEM 2.81 97.1 41 0.5 —0 TEA 0.73 98.8 42 0.5 — 0 — 0 95.8 43 0.5 — 0 — 0 95.9 44 0.5 sucrose0.5 TEM 5.62 95.5 45 0.5 sucrose 1 TEM 5.62 95.4 46 0.5 sucrose 5 TEM5.62 95.5 47 0.5 sucrose 10 TEM 5.62 95.6

Using the procedures set forth in Example 3, the formulations of Table 3are lyophilized and reconstituted with water or saline as set forth inTable 4.

TABLE 4 Light transmission properties of lyophilized MonophosphorylLipid A formulations after rehydration with water or normal saline. Wt.% of Composition after lyophilization % % % Added Diluent for % lighttransmission Sample MPL sugar Amine rehydration after rehydration 39 8.20.0 91.8 water 58.6 40 15.1 0.0 84.9 water 58.2 41 40.7 0.0 59.3 water22 42 100.0 0.0 0 water 32.8 43 100.0 0.0 0 saline 30.2 44 7.6 7.6 84.9water 63.1 45 7.0 14.0 78.9 water 64.7 46 4.5 45.0 50.5 water 50.6 473.1 62.0 34.9 water 83.5

When samples lyophilized without sugars (samples 39-43) are rehydratedwith water or saline the resultant preparation is turbid with suspendedparticulates. These samples exhibit a % transmission ranging from 22.0to 58.6. Similar results are obtained when samples 44-47 containing 7.6%to 62.0% sugar are rehydrated with water indicating that an aqueouscolloidal suspension is not formed.

EXAMPLE 5 Freezing and Thawing of Monophosphoryl Lipid A Sonicated inAqueous Triethylamine in the Presence of Sucrose

Monophosphoryl lipid A is sonicated in water containing 0.2%triethylamine (v/v) and then admixed with an equal volume of water orwith water containing added sucrose to yield a clear suspensioncontaining monophosphoryl lipid A at 0.5 mg/mL (w/v) without sucrose orcontaining 100 mg/mL sucrose w/v and triethylamine at a finalconcentration of 0.1% v/v (0.73 mg/mL w/v). The samples (48 and 49) areplaced in a Shimadzu UV-1601, UV-Visible Spectrophotometer andilluminated with light of 650 nm and each allowed 98.8% of the light topass thus indicating the formation of an aqueous colloidal suspension.The colloidal suspensions are frozen and then thawed. Upon thawing, themonophosphoryl lipid A preparation without sucrose (sample 48) is turbidwith particulates and has a % light transmission of 60.3% as measured ina Shimadzu UV-1601, UV-Visible Spectrophotometer and illuminated withlight of 650 nm wavelength indicating that an aqueous colloidalsuspension is not formed. The monophosphoryl lipid A containing sucrose(sample 49) remains clear after freezing and thawing and has a % lighttransmission of 97.8% as measured in a Shimadzu UV-1601, UV-VisibleSpectrophotometer and illuminated with light of 650 nm wavelengthindicating the formation of an aqueous colloidal suspension. These dataare displayed in Table 5.

TABLE 5 Light transmission properties before and after freezing and.thawing of Monophosphoryl Lipid A sonicated with triethylamine anddiluted with or without sucrose Composition of MPL preparations % lightAppear- Added transmission ance MPL Sucrose TEA Before After afterSample (mg/mL) (mg/mL) (mg/mL) freezing thawing thawing 48 0.5 0 0.7398.8 60.3 Turbid 49 0.5 100 0.73 98.8 97.3 Clear

EXAMPLE 6 Preparation of Vaccine Compositions

a. Preparation of Aqueous Colloidal Suspensions of Monophosphoryl LipidA

Using the procedures set forth in Example 3 above, a mixture ofmonophosphoryl lipid A in water of about 0.5 mg/ml and an amine-basedsurfactant triethanolamine at about 2.8 mg/ml is heated and sonicated togive an aqueous colloidal suspension. Either before or after sonication,but prior to freezing or lyophilizing, sucrose is added at a finalconcentration between about 10 to 200 mg/ml. The aqueous colloidalsuspension so obtained may be either frozen and thawed for use in avaccine composition or lyophilized and reconstituted with an aqueousdiluent for use in a vaccine composition.

b. Preparation of an Aqueous Vaccine Composition From FrozenMonophosphoryl Lipid A Composition

The aqueous colloidal suspension of monophosphoryl lipid A, sucrose andtriethanolamine prepared as in (a) above is frozen. It is then thawedand combined with an aqueous diluent containing an antigen, for example,a pneumococcal glycocon-jugate prepared according to U.S. Pat. No.5,360,897, to obtain a vaccine composition containing up to about 400micrograms monophosphoryl lipid A per ml and up to about 200 microgramspneumococcal glycoconjugate per ml. To obtain a vaccine compositioncontaining 400 micrograms of monophosphoryl lipid A and 200 microgramsof pneumococcal glycocongugate, for example, 0.8 ml of the thawedcolloidal suspension may be combined with 200 micrograms of pneumococcalglycocongugate in 0.2 ml of water. This vaccine composition may then beadministered to a vertebrate, preferably to a human, using about 0.1 to1.0 ml per dose.

c. Preparation of an Aqueous Vaccine Composition from LyophilizedMonophosphoryl Lipid A Composition

The aqueous colloidal suspension of monophosphoryl lipid A, sucrose andtriethanolamine prepared in (a) above is lyophilized. It is thenreconstituted with an aqueous diluent containing an antigen, forexample, a pneumococcal glyco-conjugate prepared according to U.S. Pat.No. 5,360,897, to obtain a vaccine composition containing up to about400 micrograms monophosphoryl lipid A per ml and up to about 200micrograms pneumococcal glycoconjugate per ml. This vaccine compositionmay then be administered to a vertebrate, preferably to a human, usingabout 0.1 to 1.0 ml per dose.

d. Preparation of a Frozen Aqueous Vaccine Composition

To the aqueous colloidal suspension of monophosphoryl lipid A, sucroseand triethanolamine prepared in (a) above is added an antigen, forexample, a pneumococcal glycoconjugate prepared according to U.S. Pat.No. 5,360,897 to obtain a vaccine composition. The vaccine compositionis then frozen. The concentrations of monophosphoryl lipid A andpneumococcal glycoconjugate are adjusted by addition of a aqueousdiluent, to up to about 400 micrograms per ml and up to about 200micrograms per ml, respectively, either before freezing or afterfreezing and thawing, provided that the sucrose is kept at aconcentration of about 10 to 200 mg/ml before freezing. The frozen andthawed vaccine composition may then be administered to a vertebrate,preferably to a human, using about 0.1 to 1.0 ml per dose.

e. Preparation of a Lyophilized Vaccine Composition

To the aqueous colloidal suspension of monophosphoryl lipid A, sucroseand triethanolamine prepared in (a) above is added, for example, apneumococcal glycoconjugate prepared according to U.S. Pat. No.5,360,897 to obtain a vaccine composition. The antigen may be addedeither before or after the heating and sonicating steps. The amount ofpneumococcal glycoconjugate added is calculated such that, uponsubsequent reconstitution of the lyophilized vaccine composition, theaqueous mixture will contain up to about 400 micrograms ofmonophosphoryl lipid A per ml and up to about 200 microgramspneumococcal glycoconjugate per ml. The vaccine composition is thenlyophilized. Following lyophilization, the composition is reconstitutedwith an aqueous diluent. This reconstituted aqueous vaccine compositionmay then be administered to a vertebrate, preferably to a human, usingabout 0.1 to 1.0 ml per dose.

What is claimed:
 1. A method for the preparation of an aqueous colloidalsuspension containing 3-O-desacyl-4′-monophosphoryl lipid A capable ofbeing frozen and thawed comprising: a. suspending3-O-desacyl-4′-monophosphoryl lipid A in an amount from 0.5 mg/ml up to5 mg/ml and, an amine based surfactant in an amount from 0 to about 6mg/ml in an aqueous diluent; b. forming an aqueous colloidal suspensionhaving a light transmission of greater than or equal to 88%, as measuredspectrophotometrically; c. adding sugar at about 10 to 200 mg/ml eitherbefore or after forming the aqueous colloidal suspension; d. freezingthe sugar containing aqueous colloidal suspension; and e. thawing andrecovering the aqueous colloidal suspension without sonication toprepare an aqueous colloidal suspension.
 2. The method of claim 1,wherein said thawed aqueous colloidal suspension is combined with anaqueous diluent further containing an antigen or antigens, additionaladjuvant or a pharmaceutically acceptable preservative, carrier orvehicle.
 3. The method of claim 2 wherein said additional adjuvant isaluminum phosphate.
 4. The method according to claim 2 wherein theantigen or antigens are from or produced by a bacterium, a virus, aparasite, a cancer cell or an allergen.
 5. The method according to claim4 wherein the antigen is a Chlamydia, Nontypeable Haemophilusinfluenzae, Helicobacter pylon, Moraxella catarrhalis, Neisseriagonorrhoeae, Neisseria meningitidis, Salmonella typhi, Streptococcuspneumoniae, Group A Streptococci, Group B Streptococcus, Herpes SimplexVirus, Human Immunodeficiency Virus, Human Papilloma Virus, InfluenzaVirus, Measles, Parainfluenza, Respiratory Syncytial Virus, Rotavirus,or Norwalk Virus antigen.
 6. The method according to claim 5 wherein theantigen is a conjugate comprising capsular polysaccharide ofStreptococcus pneumoniae covalently attached to a protein.
 7. The methodaccording to claim 1 the sugar comprises dextrose, mannose, galactose,fructose, sucrose, lactose, isomaltose, maltose or trehalose.
 8. Themethod according to claim 7 wherein the sugar is sucrose or dextrose. 9.The method according to claim 1 wherein the amine based surfactant istriethylamine or triethanolamine.
 10. A method for the preparation of anaqueous colloidal suspension containing 3-O-desacyl-4′-monophosphoryllipid A capable of being lyophilized and resuspended comprising: a.suspending 3-O-desacyl-4′-monophosphoryl lipid A in an amount from 0.5mg/ml up to 5 mg/ml and, an amine based surfactant in an amount from 0to about 6 mg/ml in an aqueous diluent; b. forming an aqueous colloidalsuspension having a light transmission of greater than or equal to 88%,as measured spectrophotometrically; c. adding sugar at about 10 to 200mg/ml either before or after forming the aqueous colloidal suspension;d. lyophilizing the sugar containing aqueous colloidal suspension toobtain a lyophilized composition; e. reconstituting the lyophilizedcomposition without sonication with an aqueous diluent at an amount from10 to 210 mg of the lyophilized composition per ml of aqueous diluent,and f. obtaining an aqueous composition in the form of an aqueouscolloidal suspension having a light transmission of greater than orequal to 88% as measured spectrophotometrically without sonication. 11.The method of claim 10 wherein said aqueous colloidal suspension iscombined with an aqueous diluent further containing an antigen orantigens, additional adjuvant or a pharmaceutically acceptablepreservative, carrier or vehicle.
 12. The method of claim 11 whereinsaid additional adjuvant is aluminum phosphate.
 13. The method accordingto claim 11 wherein the antigen or antigens are from or produced by abacterium, a virus, a parasite, a cancer cell or an allergen.
 14. Themethod according to claim 13 wherein the antigen is a Chlamydia,Nontypeable Haemophilus influenzae, Helicobacter pylori, Moraxellacatarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonellatyphi, Streptococcus pneumoniae, Group A Streptococci, Group BStreptococcus, Herpes Simplex Virus, Human Immunodeficiency Virus, HumanPapilloma Virus, Influenza Virus, Measles, Parainfluenza, RespiratorySyncytial Virus, Rotavirus, or Norwalk Virus antigen.
 15. The methodaccording to claim 14 wherein the antigen is a conjugate comprisingcapsular polysaccharide of Streptococcus pneumoniae covalently attachedto a protein.
 16. The method according to claim 10 wherein the sugarcomprises dextrose, mannose, galactose, fructose, sucrose, lactose,isomaltose, maltose or trehalose.
 17. The method according to claim 16wherein the sugar is sucrose or dextrose.
 18. The method according toclaim 10 therein the amine based surfactant is triethylamine ortriethanolamine.